Enhanced biogas production from nitrogen bearing feed stocks

ABSTRACT

A system comprising a first anaerobic digester ( 12 ), an ammonia recovery vessel ( 16 ) and a second anaerobic digester ( 14 ). Microorganisms in the first digester are primarily hydrolyzers and acetogens, while those in the second digester are primarily methanogens. A nitrogen containing feed stock undegoes hydrolysis ans acetogenesis in the first digester. The effluent is passed to the ammonia recovery vessel in which ammonia is removed. The low ammonia effluent stream is passed to the second digester to undergo methanogenesis, generating a biogas. In an alternate embodiment, a single anaerobic digester is used. The effluent is treated for ammonia removal and recycled to the digester to keep the ammonia levels sufficiently low to avoid ammonia inhibition.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the anaerobic treatment of feed stocksto generate biogas, and more particularly to the anaerobic treatment ofnitrogen containing feed stocks to generate a biogas.

[0002] Over the past several decades, extensive scientific andengineering work has been conducted on the biogasification of wastematerials. The fundamental technique relies on the anaerobic digestionor fermentation process. Anaerobic digestion of biomass materialsproceeds in three distinct and sequential pathways. These pathways arehydrolysis, acetogenesis and methanogenesis. The anaerobicmicroorganisms that conduct the first two steps, the hydrolyzers andacetogens, break the complex biomass molecules down into small chainmolecules. Proteins are hydrolyzed into proteoses, peptones andpolypeptides. These compounds are further broken down into ammonia andsmall chain fatty acids such as acetic acid, butyric acid, propionicacid, and lactic acid. The anaerobic microorganisms that perform thehydrolysis and acetogenesis functions are highly resistant to ammonia.Anaerobic fermentation of high nitrogen wastes using thesemicroorganisms have produced digested streams containing in excess of10,000 ppm ammonia. However, the anaerobic microorganisms responsiblefor methanogenesis are inhibited by ammonia. Methanogenesic anaerobicbacteria cease to function effectively at ammonia concentrations equalto or greater than approximately 1,200 ppm ammonia. (Kayhanian, M.,Environmental Technology, Vol. 20, PP 355-365. 1999)

[0003] Technologies such as the Upflow Anaerobic Sludge Blanket (UASB)reactor and the Extended Granular Sludge Bed (EGSB) reactor offeradvantages in the anaerobic fermentation or digestion of wastewater orother feed stocks. These reactors allow for higher treatment rates usingsmaller vessels, thereby reducing capital costs. These reactors alsoprovide for improved odor control. Still, problems associated withammonia inhibition have made these reactors relatively unstable anddifficult to operate when using feed stocks containing relatively highconcentrations of nitrogen. To mitigate these problems, it has beenproposed to control the carbon to nitrogen (C/N) ratio of the feed stockand to dilute the reactors with water in cases of sudden, large ammoniaoverloads. These proposals still suffer from a number of disadvantages.For example, adjusting the ammonia concentration in a reactor byadjusting the C/N ratio of the feed stock is a slow process, it can bedifficult to accurately determine the C/N ratio, and adjusting the C/Nratio may prove to be insufficient to handle feed stocks that are proneto generate relatively high ammonia concentrations during anaerobicdigestion. Dilution of a reactor with water also has a number ofdisadvantages. For example, diluting the reactor with water mayseriously decrease the reactor's biogas production for extended periodsof time and will typically lead to increased dewatering costs. Dilutionof an existing feed stock increases the required reactor volume fordigestion of that feed stock. An existing reactor would have a decreasedcapacity for treating a given feed stock if that feed stock werediluted.

SUMMARY OF THE INVENTION

[0004] It is therefore an object of the present invention to provide astable system for treating nitrogen containing biomass materials togenerate a biogas.

[0005] It is a further object of the present invention to provide asystem of the above type that provides for separation of the anaerobicdigestion process so that methanogenesis takes place in a separatereactor or other receptacle.

[0006] It is a still farther object of the present invention to providea system of the above type in which ammonia removal prior tomethanogenesis keeps ammonia levels sufficiently low to avoid ammoniainhibition problems.

[0007] It is a still further object of the present invention to providea system of the above type in which the system is operated undermesophilic conditions.

[0008] It is a still further object of the present invention to providea system of the above type in which the system is operated underthermophilic conditions.

[0009] It is a still further object of the present invention to providean alternate embodiment of a system of the above type in which effluentfrom a reactor is treated for ammonia removal and recycled to thereactor at a rate sufficient to keep ammonia levels within the reactorsufficiently low to avoid ammonia inhibition problems.

[0010] Toward the fulfillment of these and other objects and advantages,the system of the present invention comprises a first anaerobicdigester, an ammonia recovery vessel, and a second anaerobic digester.Microorganisms within the first anaerobic digester are primarilyhydrolyzers and acetogens, and microorganisms within the secondanaerobic digester are primarily methanogens. A nitrogen containing feedstock is passed to the first digester in which the feed stock is treatedto accomplish hydrolysis and acetogenesis. An effluent stream from thefirst digester is passed to the ammonia recovery vessel in which ammoniais removed to generate a low ammonia effluent stream. The low ammoniaeffluent stream is then passed to the second digester in which it istreated to accomplish methanogenesis, thereby generating a biogas. In analternate embodiment, a single anaerobic digester is used, an effluentstream is removed from the reactor, treated for ammonia removal, andrecycled to the digester at a rate sufficient to keep ammonia levelswithin the digester sufficiently low to avoid ammonia inhibitionproblems. The systems may be operated under mesophilic or thermophilicconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above brief description, as well as further objects, featuresand advantages of the present invention will be more fully appreciatedby reference to the following detailed description of the presentlypreferred but nonetheless illustrative embodiments in accordance withthe present invention when taken in conjunction with the accompanyingdrawings, wherein:

[0012]FIG. 1 is a schematic diagram of a system of the presentinvention; and

[0013]FIG. 2 is a schematic diagram of an alternate embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014] Referring to FIG. 1, the reference numeral 10 refers in generalto a system of the present invention, comprising two anaerobic digestersor reactors 12 and 14 and an ammonia removal or recovery vessel 16.Anaerobic microorganisms comprising primarily hydrolzyers and acetogens18 are present in the first reactor 12, and anaerobic microorganismscomprising primarily methanogens 20 are present in the second reactor14.

[0015] The structure of the reactors 12 and 14 is similar, so only onewill be described in detail. The reactor 12 features an enclosure 22defining a chamber 24. A conduit 26 is provided near the bottom of theenclosure 22 for providing a nitrogen containing feed stock. Baffles 28near the bottom help to distribute the feed stock. Above the baffles 28is a fluidized bed area 30 having the anaerobic microorganismscomprising primarily hydrolyzers and acetogens 18. A second set ofbaffles 32 is provided at an intermediate point above the fluidized bedarea 30 to serve as a first settler area. A downer conduit 34 extendsbetween the second set of baffles 32 and the lower first set of baffles28. A third set of baffles 36 is provided near an upper portion of thechamber 24 to serve as a second settler area. A gas riser conduit 38extends between the second and third sets of baffles 32 and 36. The areabetween the second and third sets of baffles 32 and 36 comprises apolishing area 40. An effluent conduit 42 exits the enclosure 22 abovethe third set of baffles 36, and a gas removal conduit 44 exits from atop portion of the enclosure 22. The conduit 42 connects the firstreactor 12 to an ammonia recovery or removal vessel or receptacle 16.Any of a wide variety of ammonia recovery or removal processes andvessels may be used. Ammonia recovery and removal processes such as theAmmonia Recovery Process, (ARP), adsorption, air stripping, steamstripping, and combinations of such processes are well known in the artand will not be discussed in detail. Conduits 46 and 48 may provide forthe introduction of reactants, such as H₂SO₄, and for the recovery orremoval of ammonia, such as in the form of (NH₄)₂SO₄. An effluentconduit 50 connects the ammonia recovery vessel 16 to the second reactor14, providing a feed stock near the bottom of the chamber 24. Thestructure of the second reactor 14 is similar to the structure of thefirst reactor 12 and will not be discussed in detail. In the fluidizedbed area 30 of the second reactor 14, the anaerobic microorganisms arecomprised primarily of methanogens 20.

[0016] As also shown in FIG. 1, a receptacle or vessel 52 may beprovided for hydrothermal liquefaction, rendering, or a similar processupstream of the first reactor 12.

[0017] Referring to FIG. 2, an alternate embodiment is disclosed inwhich effluent recycle with ammonia removal is used in connection with asingle anaerobic digestion reactor 54. The structure of the reactor 54is similar to the structure of the reactors 12 and 14 discussed aboveand will not be discussed in more detail. In the alternate embodiment, aconduit 56 exits the ammonia recovery vessel 16, and conduits 58 and 60allow for a portion of the effluent from the reactor to be recycled tothe reactor 54 after being treated for ammonia removal or recovery and aportion of that effluent to be passed for disposal or furtherprocessing. The conduit 58 connects with feed conduit 26 or enters theenclosure 22 near the bottom of the chamber 24.

[0018] Returning to FIG. 1, in operation, a nitrogen containing feedstock or waste stream containing biomass materials is fed via conduit 62to a first receptacle or vessel 52 for hydrothermal liquefaction,rendering, or a similar process. The feed stock may be a high chemicaloxygen demand (COD), high nitrogen wastewater, such as from aconcentrated animal feeding operation (CAFO). For the present system 10,it is preferred to use a feed stock stream comprising a high strengthwastewater containing a low-level of suspended solids. Such a stream isproduced by the hydrothermal liquefaction of biomass materials such assewage sludge. Such streams contain a high Total Kjeldahl Nitrogen (TKN)and ammonia nitrogen load as well as a high biological oxygen demand.The benefit from hydrothermal liquefaction is that it dissolves biomasssolids by hydrolyzing hair, cellulose, and cellular proteins intowater-soluble saccharides, proteoses, peptones and peptides. Thesewater-soluble and partially water-soluble materials are in a form thatis much more available to anaerobic microorganisms. After treatment forhydrothermal liquefaction, the stream passes via conduit 26 to a lowerportion of the first reactor 12.

[0019] In the first reactor 12, the hydrolyzers and acetogens 18 arereacted with the feed stock to accomplish hydrolysis and acetogenesis.The hydrolyzers and acetogens 18 are highly resistant to ammonia, sothese two stages of anaerobic fermentation or digestion may beaccomplished without ammonia inhibition problems even for feed stockswith relatively high nitrogen contents. For example, the concentrationof ammonia within the first reactor 12 will often reach or exceed 1,200ppm, will sometimes reach or exceed 5,000 ppm, and will occasionallyreach or exceed 10,000 ppm, all without creating ammonia inhibitionproblems for the hydrolysis and acetogenesis functions of the reactor12. The reaction is preferably accomplished under mesophilic conditionsand more preferably under thermophilic conditions. Mesophilic digestionis accomplished at a temperature that is preferably substantially withina range of from approximately 30° C. to approximately 45° C., and thatis more preferably approximately 37° C. Thermophilic digestion isaccomplished at a temperature that is preferably substantially within arange of from approximately 45° C. to approximately 60° C., and that ismore preferably approximately 55° C. This elevated temperature isbeneficial because it aids in the removal of ammonia by all three of theprocesses mentioned above. In addition, thermophilic reactions aretypically faster than mesophilic processes conducted at approximately30°-45° C. An advantage of processing high strength wastewater in athermophilic anaerobic digester is that the residence time and size ofthe reactor is smaller for a given throughput. An advantage ofprocessing high strength wastewater in a mesophilic anaerobic digesteris that the feed stock does not have to be heated to as high atemperature as needed in a thermophilic reactor.

[0020] A relatively small amount of biogas is produced in the firstreactor 12 and exits from conduit 44 at the top of the enclosure 22. Thefirst reactor 12 preferably generates less than approximately 30% of thetotal amount of biogas generated by the system 10 and more preferablygenerates less than approximately 10% of the total amount of biogasgenerated by the system 10.

[0021] An acetate and ammonia rich effluent passes from an upper portionof the first reactor 12 to the ammonia recovery vessel 16 via conduit42. In the ammonia recovery vessel 16, ammonia is recovered or removedby any known ammonia recovery or removal process. Conduits 46 and 48 mayprovide for the introduction of reactants, such as H₂SO₄, and for therecovery or removal of ammonia, such as in the form of (NH₄)₂SO₄. Otherreactants such as NaOH may be used to adjust the pH of the low ammonia,acetate rich stream to improve its suitability for methanogen digestion.Conduit 50 passes a low ammonia, acetate rich effluent stream from theammonia recovery vessel 16 to the second reactor 14. The ammonia contentin the low ammonia effluent stream is preferably below approximately1,200 ppm and is more preferably below approximately 600 ppm.

[0022] In the second reactor 14, the methanogens 20 are reacted with thelow ammonia, acetate rich feed stream to accomplish methanogenesis. Themethanogens 20, or anaerobic microorganisms responsible formethanogenesis, are inhibited by ammonia. Methanogens 20 cease tofunction effectively at ammonia concentrations equal to or greater thanapproximately 1,200 ppm ammonia. Accordingly, sufficient ammonia isremoved during the ammonia recovery stage to maintain the ammoniaconcentration within the second reactor 14 at a level that is preferablybelow approximately 1,200 ppm and more preferably below approximately600 ppm. The anaerobic methanogenesis reaction is preferablyaccomplished under mesophilic conditions and more preferably underthermophilic conditions. Mesophilic digestion is accomplished at atemperature that is preferably substantially within a range of fromapproximately 30° C. to approximately 45° C., and that is morepreferably approximately 37° C. Thermophilic digestion is accomplishedat a temperature that is preferably substantially within a range of fromapproximately 40° C. to approximately 60° C., and that is morepreferably approximately 55° C. This elevated temperature is beneficialbecause it aids in the removal of ammonia by all three of the processesmentioned above. In addition, thermophilic reactions are typicallyfaster than mesophilic processes conducted at approximately 30°-45° C.

[0023] A relatively large amount of biogas is produced in the secondreactor 14 and exits from the top of the enclosure 22 via conduit 44.The second reactor 14 preferably generates greater than approximately70% of the total amount of biogas generated by the system 10 and morepreferably generates greater than approximately 90% of the total amountof biogas generated by the system 10. A low COD, low ammonia effluent isdischarged from an upper portion of the second reactor 14 via conduit 64and may be passed to a wastewater treatment process or facility.

[0024] In this manner, the anaerobic fermentation is conducted in twoseparate stages. The hydrolysis and/or acetogenesis portion of thefermentation is conducted in a first stage, the ammonia is then removedfrom the acetate rich wastewater, and methanogenesis is conducted in thesecond stage of the anaerobic fermentation. This allows for superiorbiogas production levels while avoiding problems associated with ammoniainhibition.

[0025] In the alternate embodiment depicted in FIG. 2, a reactor orupflow anaerobic digester 54 such as an EGSB is provided with an ammoniaremoval vessel 16 and a recycle stream 58. A nitrogen containing feedstock or waste stream containing biomass materials may be fed to a firstreceptacle or vessel (not shown in FIG. 2) for hydrothermalliquefaction, rendering, or a similar process. The feed stock may be ahigh COD, high nitrogen wastewater, such as from a CAFO. After treatmentfor hydrothermal liquefaction, the stream passes via conduit 26 to alower portion of the reactor 54.

[0026] In the reactor 54, the hydrolyzers, acetogens, and methanogens 18and 20 are fed with the feed stock to accomplish the hydrolysis,acetogenesis, and methanogenesis reactions. The reaction is preferablyaccomplished under mesophilic conditions and more preferably underthermophilic conditions. Mesophilic digestion is accomplished at atemperature that is preferably substantially within a range of fromapproximately 30° C. to approximately 45° C., and that is morepreferably approximately 37° C. Thermophilic digestion is accomplishedat a temperature that is preferably substantially within a range of fromapproximately 45° C. to approximately 60° C., and that is morepreferably approximately 55° C.

[0027] Biogas is produced in the reactor 54 and exits from the top ofthe enclosure 22 via conduit 44. An effluent passes from an upperportion of the reactor to the ammonia recovery vessel 16 via conduit 42.In the ammonia recovery vessel 16, ammonia is recovered or removed byany known ammonia recovery or removal process. Conduits 46 and 48 mayprovide for the introduction of reactants, such as H₂SO₄, and for therecovery of ammonia, such as in the form of (NH₄)₂SO₄. Other reactantssuch as NaOH may be used to adjust the pH of the low ammonia, acetaterich stream to improve its suitability for methanogen digestion. The lowammonia effluent exits the ammonia recovery vessel 16 via conduit 56,and a portion of the low COD, low ammonia stream may be passed viaconduit 60 to a wastewater treatment process or facility. Conduit 58passes a portion of the low ammonia effluent stream from the ammoniarecovery vessel 16 to the reactor 54 to dilute the feed stock so thatammonia levels in the reactor 54 remain below inhibition levels. Theammonia content in the low ammonia effluent stream is preferably belowapproximately 1,200 ppm, is more preferably below approximately 600 ppm,and is most preferably below approximately 300 ppm. Similarly, becausemethanogens 20 are in the reactor 54, it is important to maintain arelatively low ammonia concentration within the reactor 54. A sufficientdegree of ammonia removal is achieved in the ammonia recovery vessel 16,and a sufficient portion of the low ammonia effluent stream is recycledto the reactor 54 to maintain ammonia concentrations within the reactor54 at a level that will avoid ammonia inhibition problems. The ammoniaconcentration within the reactor 54 is preferably maintained belowapproximately 1,200 ppm and is more preferably maintained belowapproximately 600 ppm.

[0028] The configuration depicted in FIG. 2 may be used when the feedmaterial is highly concentrated or contains toxic materials. Asdiscussed in more detail above, in this embodiment, ammonia iscontinuously removed from a recycle stream, which enables the reactor 54to operate at a sufficiently low ammonia concentration such that themethanogens 20, or methanogenic bacteria, are not inhibited. Similar tothe first embodiment, the ammonia is removed by use of an AmmoniaRecovery Process, ARP, adsorption, air stripping, or steam stripping, orany of a wide variety of ammonia removal or recovery techniques or somecombination thereof.

[0029] The ammonia inhibition of methanogenic anaerobic bacteria byammonia is a limitation on the production of biogas from nitrogencontaining feed stocks. It will be appreciated that the present system10 may be incorporated into or used in connection with a wide variety ofanaerobic digestion systems and technologies, such as UASB and EGSBsystems, to improve biogas production.

[0030] Other modifications, changes and substitutions are intended inthe foregoing, and in some instances, some features of the inventionwill be employed without a corresponding use of other features. Forexample, the system 10 may be used with or without wastewaterpretreatment such as hydrothermal liquefaction, rendering or the like.Also, the feed stock may come from any number of different sources,including but not limited to CAFOs, food processing plants, andwastewater treatment facilities. Further, the recycle feature may beused in connection with either embodiment. Further still, it isunderstood that the reactor receptacles or vessels 12, 14, 16, and 54may take any number of shapes, sizes, and configurations and need nothave the structure described in connection with the preferredembodiments. Further, it is understood that the various reactions maytake place under different conditions, using different styles or typesof reactors. For example, it is understood that hydrolysis andacetogenesis may be accomplished under thermophilic conditions whilemethanogenesis may be accomplished under mesophilic conditions, and viceversa. Similarly, it is understood that hydrolysis and acetogenesis maybe accomplished using a reactor such as an UASB reactor whilemethanogenesis may be accomplished using a different style reactor suchas an EGSB reactor, or vice versa. It is also understood that allquantitative information given is by way of example only and is notintended to limit the scope of the present invention. Accordingly, it isappropriate that the appended claims be construed broadly and in amanner consistent with the scope of the invention.

What is claimed is:
 1. A method, comprising: (1) passing a nitrogencontaining feed stock to a first receptacle; (2) treating said feedstock in said first receptacle to accomplish hydrolysis andacetogenesis; (3) after step (2), passing said feed stock to a secondreceptacle; (4) treating said feed stock in said second receptacle toremove ammonia from said feed stock to generate a low ammonia effluentstream; (5) passing said low ammonia effluent stream to a thirdreceptacle; and (6) treating said low ammonia effluent stream in saidthird receptacle to accomplish methanogenesis.
 2. The method of claim 1,wherein step (2) comprises: treating said feed stock in said firstreceptacle to accomplish hydrolysis and acetogenesis by anaerobicfermentation during which said feed stock reaches a first level ofammonia concentration that is equal to or greater than approximately1,200 ppm.
 3. The method of claim 1, wherein step (2) comprises:treating said feed stock in said first receptacle to accomplishhydrolysis and acetogenesis by anaerobic fermentation during which saidfeed stock reaches a first level of ammonia concentration that is equalto or greater than approximately 5,000 ppm.
 4. The method of claim 2wherein: steps (2) and (6) generate an amount of a biogas; and step (2)generates less than approximately 30% of said amount of said biogas. 5.The method of claim 2 wherein: steps (2) and (6) generate an amount of abiogas; and step (2) generates less than approximately 10% of saidamount of said biogas.
 6. The method of claim 5, wherein step (4)comprises treating said feed stock in said second receptacle to removeammonia from said feed stock to generate a low ammonia effluent streamcomprising an ammonia concentration of less than approximately 1,200ppm.
 7. The method of claim 5, wherein step (4) comprises treating saidfeed stock in said second receptacle to remove ammonia from said feedstock to generate a low ammonia effluent stream comprising an ammoniaconcentration of less than approximately 600 ppm. 8.The method of claim6, wherein: step (4) further comprises treating said feed stock in saidfirst receptacle at a temperature substantially within a range of fromapproximately 45° C. to approximately 60° C.; and step (6) furthercomprises treating said low ammonia effluent stream in said thirdreceptacle at a temperature substantially within a range of fromapproximately 45° C. to approximately 60° C.
 9. The method of claim 6,wherein: step (4) further comprises treating said feed stock in saidfirst receptacle at a temperature substantially within a range of fromapproximately 30° C. to approximately 45° C.; aid step (6) furthercomprises treating said low ammonia effluent stream in said thirdreceptacle at a temperature substantially within a range of fromapproximately 30° C. to approximately 45° C.
 10. The method of claim 5,further comprising: treating said feed stock to accomplish hydrothermalliquefaction before passing said feed stock to said first receptacle.11. A method, comprising: (1) passing a nitrogen containing feed stockto a first receptacle; (2) treating said feed stock in said firstreceptacle to accomplish hydrolysis, acetogenesis, and methanogenesis;(3) removing a biogas generated in said first receptacle; (4) passing aneffluent from said first receptacle to a second receptacle; (5) treatingsaid effluent in said second receptacle to remove ammonia from saideffluent to leave a low ammonia effluent; and (6) passing an amount ofsaid low ammonia effluent to said first receptacle.
 12. The method ofclaim 11, wherein step (6) comprises: passing an amount of said lowammonia effluent to said first receptacle, said amount being sufficientto maintain an ammonia concentration within said first receptacle ofless than approximately 1,200 ppm.
 13. The method of claim 11, whereinstep (6) comprises: passing an amount of said low ammonia effluent tosaid first receptacle, said amount being sufficient to maintain anammonia concentration within said first receptacle of less thanapproximately 600 ppm.
 14. The method of claim 12, further comprising:maintaining contents within said first receptacle at a temperature thatis substantially within a range of from approximately 45° C. toapproximately 60° C.
 15. An apparatus, comprising: a first vesselcomprising an anaerobic digester; a first amount of active anaerobicmicroorganisms within said first vessel comprising primarily hydrolyzersand acetogens; a second vessel comprising an ammonia removal vessel,said second vessel being operably connected to said first vessel toreceive a first effluent from said first vessel; a third vesselcomprising an anaerobic digester, said third vessel being operablyconnected to said second vessel to receive a second effluent from saidsecond vessel; and a second amount of active anaerobic microorganismswithin said third vessel comprising primarily methanogens.
 16. Theapparatus of claim 15, wherein said first and third vessels compriseupflow anaerobic sludge blanket reactors.
 17. The apparatus of claim 15,wherein said first and third vessels comprise extended granular sludgebed reactors.
 18. The apparatus of claim 15, further comprising a fourthvessel for conducting hydrothermal liquefaction, said first vessel beingoperably connected to said fourth vessel to receive a third effluentfrom said fourth vessel.
 19. The apparatus of claim 15 wherein saidhydrolyzers and said acetogens comprise at least approximately 70% ofsaid first amount of said active anaerobic microorganisms.
 20. Theapparatus of claim 15 wherein said methanogens comprise at leastapproximately 70% of said second amount of said active anaerobicmicroorganisms.
 21. The apparatus of claim 19 wherein said methanogenscomprise at least approximately 70% of said second amount of said activeanaerobic microorganisms.